Biochemical analysis of tumor rejection antigen and its role of oncogenesis.

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The antigen peptides recognized by RL*1 specific CTL were determined by direct sequencing of the fraction separated by reversed-phase high performance liquid chromatography. Molecular analysis revealed the antigen peptide were derived from the 5'-untransrated region of proto-oncogene c-akt by insertion of a murine leukemia virus LTR together with six nucleotides of unknown origin. In this study, we investigated this altered Akt molecule and its role of oncogenesis in RL*1.1. Western blot analysis using plyclonal rabbit anti c-Akt synthetic polypeptide antibodies revealed a protein 59 KD in addition to 56 KD normal c-Akt protein in RL*1 cells. 2. The 59 KD altered Akt molecule (RL-Akt) was expressed at about ten times level to normal c-Akt and was detected in the membrane fraction and in the cytosolic fraction, although the c-Akt was detected only in the cytosolic fraction. 3. RL-akt transgeneic NIH 3T3 cells grew well, forming colonies in soft apr. 4. RL-Akt deletion mutant and RL-Akt weakly expressed mutant were established. 5. To assess the effect of RL-Akt in the growth rate in vivo, these mutants and parental RL*1 cells transplanted in BALB/c mice, respectively, parental RL*1 cells grow rapidlly, but weakly expressed mutant grow slowly, and deletion mutant did more slowly.These observations suggests that the over expression and localization in the membrane of RL-Akt resulted in effective signal transduction of growth factor-PI3 pathway and these participates in malignancy in RL*1 leukemia. The study of the effects of RL-Akt to the host immune system are going now.